The overall objectives of this proposal are to improve production, purification and design of helper-dependent adenoviral (HD-Ad) vectors for their safe use in the treatment of liver disease by somatic gene therapy. Specifically, we will generate new production cell lines that are designed to allow high-titer production of HD-Ad vectors without the generation of replication competent adenoviruses (RCA). We will construct new helper viruses with a reduced chance of recombination with the DNA of the production cell line and with the vector DNA. We will establish standard procedures for large-scale and RCA-free HD-Ad vector production and we will establish chromatographic vector purification schemes. Further, we will identify stuffer DNAs that will have increased stability during production and we will improve expression of the therapeutic genes by incorporating matrix associated regions (MARs) into the vectors. We will investigate the interaction of adenoviral vectors with serum and cellular components of the innate immune system and we will develop technologies that result in efficient and safe gene transfer into the liver following intravenous vector injection. Finally, we will evaluate in appropriate animal models frequencies of vector integration into the host genome in vivo. As soon as new know-how and materials are generated they will be transferred to the members of this consortium and to the Baylor vector production core.